In this proposal, we return to the "ugly" aspect of Mycobacterium tuberculosis, to the "armor coat," the "waxy layer," i.e., the mycolates, the mycocerosates, the sulfatides, the cord factors, and the arabinosides of arabinogalactan and lipoarabinomannan, that lend the organism and the disease such uniqueness among prokaryotes and infections. We have assembled a unique consortium that can address from a modern perspective the dominant surface components of M. tuberculosis that underlie mechanisms of pathogenesis, immune-evasion, virulence and persistence. The development of novel cloning vectors for introducing recombinant DNA into Mycobacterium spp., our comprehensive understanding of the finite biochemical structure of the molecules that comprise the cellular envelope, and our ability to isolate and characterize well defined cell wall mutants now allow a novel approach to understanding disease function. Thus the central theme of this proposal is to isolate mutants deficient in major cell wall structures and to use them in definition of biosynthetic genes and biological functions. Specifically, cell wall mutants will be generated by chemical or transposon mutagenesis. Also, spontaneous mutants will be selected by screening of clinical isolates, and the formation of spontaneous mutants will be "forced" by growth of M. tuberculosis under stringent nutrient conditions. Mutants will be selected by a variety of protocols such as antibiotic sensitivity, colony morphology, radiolabeling, and probing with monoclonal antibodies and lectins. Once specific defects are chemically characterized, the genes responsible for the synthesis of the deficient entities will be defined by complementation with genes from virulent M. tuberculosis, and stable well-defined mutants will be constructed using gene replacement methodologies. Finally, these specific cell wall defined mutants will provide the means by which to measure the contribution of major cell wall components to phagocytosis, stimulation of macrophage, release of cytokines, and activation of the host cell to kill the bacterium in well established animal models. The combined abilities of this group now provide the means to solve the long- time need for the generation, characterization and evaluation of cell-wall deficient variants of M. tuberculosis.